C.P. Winlove

Oxygen and lactate concentrations measured in vivo in the intervertebral discs of patients with scoliosis and back pain.

Study Design. Oxygen concentrations in intervertebral discs were measured in 10 patients during discography and in 13 patients with scoliosis and 11 patients with back pain during spinal surgery. Lactate concentration profiles were measured in 12 of these discs. The discs were graded for degeneration by magnetic resonance maging and histology where possible. Objectives. To determine if oxygen and lactate levels in human discs vary with degree of degeneration. Failure of nutrient transport is thought to lead to disc degeneration. Summary of Background Data. The disc is avascular. Oxygen is used by the disc cells, and lactate is produced. Low oxygen and high lactate concentrations have been measured in the center of healthy animal discs. Methods. Oxygen concentrations were measured amprometrically. The sterilized gold‐needle electrode was introduced into the disc during discography or after the disc was exposed surgically via an anterior approach. Concentration profiles of each disc took approximately 5 minutes to measure. Lactate concentrations were measured biochemically on the excised disc segment. Results. Oxygen concentrations were highest at the disc surface and fell toward the center. Lactate concentrations showed the reverse profile. Oxygen levels were very variable, ranging from 5‐150 mm Hg in the center of the nucleus. No correlation was seen with age, pathology, or degree of degeneration. Lactate concentrations ranged for the most part from 2 mmol/L to 6 mmol/L. Conclusions. Concentrations of metabolites depend on cellular activity and on transport of the metabolite between the blood supply and the cell. The correlation between degeneration and nutrition cannot be determined only from metabolite concentrations; measurements of metabolic activity and nutrient transport rates also are required.

Interactions of elastin and aorta with sugars in vitro and their effects on biochemical and physical properties

SummaryStiffening of blood vessel walls occurs in the early stages of atherosclerosis, and this process is known to occur earlier in diabetic subjects. The effect could be due, in part, to glycation. Although collagen is responsible for ensuring the ultimate tensile strength of the tissue, elastin largely determines the compliance of the vessel wall in the normal physiological range of pressures and this appears to be closely matched to haemodynamic requirements. Changes in elastin are therefore likely to affect optimal function of the tissue. We have investigated the susceptibility of elastin to glycation and effects of glycation on its mechanical and physicochemical properties. We found that purified elastin and a collagen-elastin preparation from the porcine thoracic aorta rapidly incorporated glucose and ribose, the extent increasing linearly with increasing concentration and reaching a maximum after 7 days at 37°C. Biochemical analysis showed that about one of the five lysines available per elastin monomer was glycated after 12 days incubation at a sugar concentration of 250 mmol/l. In long-term incubations glycation was associated with the appearance of the advanced glycation end products, the fluorescent cross-link pentosidine and the non-fluorescent putative cross-link NFC-1. In both purified elastin and the whole elastin-collagen matrix the slope of the force-extension curve increased significantly with glycation. The greatest increase in stiffness was observed in the elastin-collagen preparation after ribose incubation (250 mmol/l for 1 month), where the slope, at large strain, increased by 56±19 % (mean±SD,n=12). The diameter of the tissue at 1 N force also changed: for elastin there was an increase in length of approximately 5 %, but for the elastin-collagen there was a decrease of similar magnitude indicating that glycation introduces differential strains within the fibrous protein matrix. Potentiometric titration demonstrated that glycation was associated both with loss of basic groups and shifts in pK of the acidic groups, which indicated changes in the environment of the charge groups due to conformational rearrangements. Changes in ion binding were dependent on pH, and were consistent with a reduction in effective anionic charge. Calcium binding to elastin was increased at acid pH, but decreased at higher pH. We suggest that these effects are not only due to changes in the charge profile, but also in the conformation of the molecule resulting from glycation of the charged lysine and arginine side-chain residues.

Focal and Regional Variations in the Composition of the Glycocalyx of Large Vessel Endothelium

The glycocalyx of the endothelium of the systemic arteries and vena cava of the rabbit was visualised by in situ perfusion fixation with glutaraldehyde containing Alcian blue. The thickness of the layer ranged from 45 +/- 1 nm in the coronary artery to 81 +/- 2 nm in the carotid. The glycocalyx was 20 +/- 1.5 nm thicker on the downstream side of intercostal ostia than on the upstream side. Changes in the staining pattern with increasing concentrations of MgCl2 indicated that carboxyl groups made the major contribution to the surface charge, though sulphate groups were also present, particularly in the aortic arch and carotid artery. Segments of the thoracic aorta and carotid artery were also stained in vitro with fluorescence labelled wheat germ agglutinin, and fluorescence intensity in histological sections was quantified using a video microscope equipped with a microcomputer-based image analysis system. The fluorescence intensity in the carotid was 1.65 +/- 0.15 times that in the aorta. Pretreatment with neuraminidase reduced fluorescence intensity by 60 +/- 4% in the carotid and 53 +/- 2% on the upstream side of intercostal ostia, but only by 37 +/- 3% on the downstream side. Chondroitinase and heparanase both reduced binding and when used together their effect was additive, reducing fluorescence by 27 +/- 3, 51 +/- 4, and 32 +/- 3% at the three sites, respectively. Though the interpretation of the lectin binding experiments is complicated by a number of factors, these results support previous reports that sialyl groups are abundant in the endothelial glycocalyx. Glycosaminoglycans are also present, however, in significant amounts.

Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer.

Study Design. An in vivo study measuring nitrous oxide concentrations in scoliotic intervertebral discs during surgery. Objectives. To determine pathways for nutrient transport into scoliotic human discs in vivo. Summary of Background Data. The intervertebral disc is the largest avascular structure in the body. Disc cells in the nucleus rely on the blood supply from the vertebral bodies for supply of nutrients and removal of waste. Loss of nutrient supply is thought to lead to disc degeneration, but solute transport has not been measured in vivo in humans. Methods. We measured solute transport into the disc using N2O as a tracer, in 19 human discs from five patients with neuromuscular scoliosis (6–19 years of age) during surgery for correction of scoliotic deformities. During anesthesia N2O diffuses into the disc at a rate governed by effective permeability of the vertebral body–disc interface. Intradiscal N2O concentrations were measured amperometrically using silver needle microelectrodes, which were inserted into the discs once they were exposed by an anterior approach. Results. For all spines N2O concentrations were very low in the disc at the curve apex (6% those expected from unimpeded diffusion) and, although still low, were significantly higher 2 discs below or above the apex. Conclusions. Because flux into the apical disc is most restricted, the decrease in solute transport is possibly induced by changes in mechanical stress on the disc; microfocal radiographs of a scoliotic spine suggest that increased endplate calcification could be partly responsible for limiting solute diffusion.

Synovial Hypoxia as a Cause of Tendon Rupture in Rheumatoid Arthritis

PURPOSE Hypoxia and angiogenesis are now recognized as being important events in the perpetuation of joint destruction in rheumatoid arthritis (RA). In 50% of patients with RA, however, the disease also involves inflammation of the synovial tissue surrounding the tendons, which is associated with multiple ruptures and poor prognosis for long-term hand function. The aim of this study was to determine whether hypoxia and angiogenesis may also play a role in RA tendon disease. METHODS Matched in vivo synovial oxygen measurements (invasive and encapsulating tenosynovium and joint synovium) were taken intraoperatively using a microelectrode technique in patients having elective hand surgery for RA. Patients having elective hand surgery for indications other than inflammatory synovitis were recruited as controls. In parallel, RA synovial tissue was harvested and stained for vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-2alpha. Tissue was also cultured under either hypoxic (1% O(2)) or normoxic (21% O(2)) conditions to investigate the effect of hypoxia on the expression of VEGF and its soluble receptor, as well as on the key cytokines interleukin (IL)-6, IL-8, IL-10 and the chemokine monocyte chemoattractant protein-1. RESULTS Invasive tenosynovium was observed to be significantly more hypoxic than either noninvasive tenosynovium or joint synovium in the same patients. Furthermore, RA tenosynovium was shown to be more hypoxic than tenosynovium in patients without RA. This hypoxia was accompanied by expression of VEGF and hypoxia-inducible factor-2alpha. Using in vitro joint synovial cell cultures, upregulation of VEGF expression was shown to be a consequence of this in vivo hypoxia. Furthermore, hypoxia downregulated release of monocyte chemoattractant protein-1 and the immunoregulatory cytokine IL-10. CONCLUSIONS These data demonstrate that hypoxia is a feature of rheumatoid tendon disease and differentially regulates angiogenesis and the inflammatory cascade in RA.

The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix.

Adipose tissue (AT) expansion in obesity is characterized by cellular growth and continuous extracellular matrix (ECM) remodeling with increased fibrillar collagen deposition. It is hypothesized that the matrix can inhibit cellular expansion and lipid storage. Therefore, it is important to fully characterize the ECMs biomechanical properties and its interactions with cells. In this study, we characterize and compare the mechanical properties of human subcutaneous and omental tissues, which have different physiological functions. AT was obtained from 44 subjects undergoing surgery. Force/extension and stress/relaxation data were obtained. The effects of osmotic challenge were measured to investigate the cellular contribution to tissue mechanics. Tissue structure and its response to tensile strain were determined using nonlinear microscopy. AT showed nonlinear stress/strain characteristics of up to a 30% strain. Comparing paired subcutaneous and omental samples (n = 19), the moduli were lower in subcutaneous: initial 1.6 ± 0.8 (means ± SD) and 2.9 ± 1.5 kPa (P = 0.001), final 11.7 ± 6.4 and 32 ± 15.6 kPa (P < 0.001), respectively. The energy dissipation density was lower in subcutaneous AT (n = 13): 0.1 ± 0.1 and 0.3 ± 0.2 kPa, respectively (P = 0.006). Stress/relaxation followed a two-exponential time course. When the incubation medium was exchanged for deionized water in specimens held at 30% strain, force decreased by 31%, and the final modulus increased significantly. Nonlinear microscopy revealed collagen and elastin networks in close proximity to adipocytes and a larger-scale network of larger fiber bundles. There was considerable microscale heterogeneity in the response to strain in both cells and matrix fibers. These results suggest that subcutaneous AT has greater capacity for expansion and recovery from mechanical deformation than omental AT.

The distributions and diffusivities of small ions in chondroitin sulphate, hyaluronate and some proteoglycan solutions

Abstract The distributions and diffusivities of Na + , Ca 2+ and Cl − in chondroitin sulphate (CS), hyaluronate (HA) and proteoglycan solutions were measured using equilibrium dialysis and a capillary tube method. Measurements were made for a range of glycosaminoglycan (GAG) concentrations up to those normally found in dense connective tissue (10% CS, 2.5% HA), ionic strengths up to normal physiological concentrations (0.15 M) and for different combinations of monovalent and divalent cations. The partition coefficients, K i , of the positive ions increased with increasing matrix concentration and with decreasing ionic strength but with one exception the selectivity coefficient K Na Ca = √ K Ca / K Na was close to unity, indicating nearly ideal Donnan distributions. The ionic diffusivities decreased very much like those of small neutral solutes with increasing matrix concentration and with one exception were relatively independent of ionic strength, The exception in both cases was low matrix concentrations and low ionic strengths for which the diffusivity of Ca 2+ was an order of magnitude lower and selectivity coefficients were ≈ 2. We conclude that at physiological ionic strengths and GAG concentrations the distributions of small ions are determined by simple electrostatic interactions, without binding or condensation, and the diffusivities are not affected by the electrostatic field.

Alpha-1-Acid (AAG, Orosomucoid) Glycoprotein: Interaction with Bacterial Lipopolysaccharide and Protection from Sepsis

In the acute phase response to a variety of insults a rise in the levels of the acute phase proteins, including elevations of serum α1 acid glycoprotein (AAG) occurs. The physiological role of AAG is unknown, however, the time course of AAG production in the acute phase response together with its strong affinity for basic compounds suggests that AAG may function as an immune modulator to bind both exogenous and endogenous inflammatory mediators. Using E. coli lipopolysaccharide (LPS), an initiator of the acute inflammatory response associated with septic shock, we demonstrate that AAG-LPS complexes can activate mouse macrophages in vitro. In a mouse animal model of sepsis, AAG was shown to protect against meningococcal endotoxin.To pursue the mechanism of AAG action we demonstrated that AAG interacts directly with LPS using dynamic light scattering particle sizing and particle mobility. We also determined the enthalpy of interaction of AAG and LPS and showed that AAG leads to agglutination of LPS impregnated rabbit red blood cells.These studies suggest that AAG may function as an immune-modulator in the acute phase response, possibly by counter-regulating the activity of macrophage pro-inflammatory cytokines.

The measurement of oxygen diffusivity and concentration by chronoamperometry using microelectrodes

The transient response of a bare Pt microelectrode to the sudden application of the polarizing voltage is shown to be determined by the diffusivity and concentration of O2 in the surrounding medium. Using recently developed theoretical solutions, experimental measurements of the transient current are analysed to give both diffusivity and concentration in media for which independent data are available. It is shown that absolute measurements are possible but require knowledge about the electrode dimensions and about the reduction reaction which is not generally available. Measurements relative to an appropriate reference medium, on the other hand, are relatively easy and reliable. Results obtained in concentrated solutions of albumin indicate that measurements of O2 diffusivity and concentration can be made in the presence of surface-active macromolecules and suggest that this technique could be used in biological tissues.

Net albumin transport across the wall of the rabbit common carotid artery perfused in situ

We have studied the transport of radioactively labelled albumin in the rabbit common carotid artery perfused in situ at 100 cm H2O luminal pressure in the anaesthetized living animal, assessing the distribution of concentration across the wall by means of sequential frozen sectioning. We have compared the findings with those of experiments in which we have attempted to saturate the wall with label. Our findings support the belief that there is a net transport of macromolecules across the arterial wall. They show in addition that the wall is inhomogeneous. The distribution volume for label is greater in the adventitia than the media, which appears to offer a larger resistance. The transport process is seemingly dominantly diffusional.